Two kit formats are available:
- The QuantiFast Pathogen RT-PCR +IC Kit for detection of viral RNA, which contains an internal RNA control, plus the corresponding internal control primer/probe set.
- The QuantiFast Pathogen PCR +IC Kit for detection of viral, bacterial, or fungal DNA, which contains an internal DNA control, plus the corresponding internal control primer/probe set.
- With both kits, ROX is supplied in 2 tubes of different concentrations, enabling use on any real-time instrument.
The supplied 5x master mix contains HotStarTaq Plus DNA Polymerase and dNTP mix. Two separate vials of ROX dye solution enable use with real-time cyclers that require ROX reference dye in the amplification reaction, as well as those that do not. The QuantiTect Nucleic Acid Dilution Buffer stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips. The QuantiFast Pathogen +IC Kits provide a ready-to-use Internal Control for universal use as an amplification control with different pathogen assays. For addition of the Internal Control to the purification procedure, a highly concentrated Internal Control DNA (High conc.) or Internal Control RNA (High conc.) can be ordered separately.
The QuantiFast Pathogen RT Mix supplied with the QuantiFast Pathogen RT-PCR +IC Kit contains a unique formulation of Sensiscript Reverse Transcriptase optimized for sensitive detection of viral RNA.
For sensitive and reliable detection of viral RNA or DNA and bacterial DNA including an internal control
- Simultaneous detection of pathogen target and QIAGEN Internal Control
- 5x master mix for higher sensitivity with more sample input
- Easy setup of new pathogen assays with increased process safety through correct interpretation of negative results
- Clear detection of weak positive target signals
- Fast, universal protocol on real-time cyclers
QuantiFast Pathogen +IC Kits provide sensitive and rapid real-time PCR or one-step RT-PCR detection of pathogen nucleic acids using sequence-specific probes. To enable high process safety through the correct interpretation of negative results, each kit contains reagents for duplex real-time detection of a user-defined pathogen target with the QIAGEN Internal Control. For simplified use, the QIAGEN Internal Control assay design eliminates the need for optimization of duplex detection with the target. The use of one universal exogenous Internal Control for all pathogen assays allows for the parallel read-out of different pathogen targets (e.g., viral, bacterial, or fungal) and for easy implementation of new pathogen assays.
QuantiFast Pathogen +IC Kits enable simultaneous detection of viral RNA and/or DNA targets plus internal control over a wide linear range without loss of sensitivity when duplexing.
The protocol has been developed to run fast cycling experiments on most cyclers with high reliability.
- 5x QuantiFast Pathogen Master Mix Highly concentrated master mix enables larger volumes of template to be added to the assay for increased sensitivity. Optimized for sensitive pathogen detection.
- QIAGEN Internal Control (IC) Internal control templates: internal control DNA in the PCR Kit or internal control RNA in the RT-PCR Kit.
- Internal Control Assay for IC Premixed primer/probe set (TaqMan probe) labeled with MAX (equivalent to HEX, VIC, etc.).
- QuantiFast Pathogen RT Mix* Contains a unique formulation of Sensiscript Reverse Transcriptase optimized for highly sensitive detection of pathogen RNA.
- ROX Dye Solution Separate tube of passive reference dye for normalization of fluorescent signals on Applied Biosystems 7500 real-time PCR systems. Optional: Can be used on Stratagene instruments from Agilent.
- High-ROX Dye Solution Separate tube of passive reference dye for normalization of fluorescent signals on Applied Biosystems 7900 and StepOne real-time PCR systems.
- QuantiTect Nucleic Acid Dilution Buffer Proprietary buffer formulation for dilution and storage of nucleic acid standards.
- TE buffer For reconstitution of lyophilized internal control assay.
- QuantiFast Pathogen Buffer Unique buffer composition, which includes synthetic Factor MP (see Figure 'Unique Buffer Promotes Stable and Efficient Primer and Probe Annealing') to facilitate multiplex PCR, and Q-Bond to enable fast cycling. Balanced combination of K+/NH4+ ions promotes specific primer and probe annealing. No ROX included in the master mix.
- HotStarTaq Plus DNA Polymerase (included in the master mix) Activated by a 5-minute, 95°C incubation step, providing a highly stringent hot start for high sensitivity and specificity. For convenience, the master mix can be stored at 2-8°C for up to 2 months. This allows faster reaction setup by avoiding the need to thaw reaction components.
- dNTPs (included in the master mix) Mix of dATP, dCTP, dGTP, and dTTP.
The QuantiFast Pathogen +IC Kits provide a ready-to-use exogenous Internal Control for universal use with different pathogen assays, for easy setup of new targets and no need for optimization. To ensure correct interpretation of negative results, the Internal Control is detected in the same tube with the pathogen target in a duplex PCR or RT-PCR to test for successful amplification and exclude, e.g., the presence of PCR inhibitors. The premixed Internal Control Assay provided contains a primer-probe set for detection of the exogenous DNA or RNA Internal Control and has been optimized to prevent interference with target primers.
Each QuantiFast Pathogen +IC Kit includes the Internal Control Assay and Internal Control DNA or RNA for use as an amplification control via direct addition to the reaction mix. Alternatively, the Internal Control can be added to the purification procedure to control both the purification process and amplification. For addition of the Internal Control to the purification procedure, a highly concentrated Internal Control DNA or RNA (High conc.) can be ordered separately.
Combined amplification of pathogen target and Internal Control using the QuantiFast Pathogen +IC Kits increases process safety of the pathogen detection workflow by ensuring the correct interpretation of negative results.
QuantiTect Nucleic Acid Dilution Buffer supplied with the kits stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips.
The buffer enables reliable dilution of standards used to quantify viral nucleic acids, giving a wide linear range, from low to high CT values. The buffer ensures longer storage of standards without degradation.
QuantiFast Pathogen +IC Kits provide a simple procedure for detection of a user-defined pathogen and the QIAGEN Internal Control. They contain a ready-to-use master mix for the real-time detection of viral RNA (1-step RT-PCR) or viral, bacterial, and fungal DNA (PCR). There is no need for optimization of reaction and cycling conditions. Simply mix the supplied master mix with the pathogen assay (primers and probe) and the supplied Internal Control Assay and the Internal Control DNA or RNA. Alternatively, if the internal control has been added to the sample purification procedure, add RNase-free water instead of Internal Control DNA or RNA to the reaction mix. Then add your sample DNA or RNA and start the reaction on any cycler. The kit handbook contains optimized protocols for use with TaqMan probes on a wide range of cyclers. It also contains recommendations for selection of dye combinations.
Each kit is supplied with a master mix that is free of ROX dye. The kit includes 2 separate vials of ROX dye solutions of different concentrations, which can be added to reactions, depending on the real-time cycler used.
The master mix supplied with the QuantiFast Pathogen RT-PCR +IC Kit does not contain ROX dye and can be used directly with all instruments that do not require ROX dye for fluorescence normalization. Each QuantiFast Pathogen RT-PCR +IC Kit also includes two separate vials of ROX dye solutions of different concentrations, which can be added to reactions, depending on the real-time cycler used.
QuantiFast Pathogen +IC Kits provide sensitive real-time PCR or one-step RT-PCR using sequence-specific probes for detection of pathogen DNA and/or RNA including an Internal Control. The kits can be used on a wide range of real-time cyclers from QIAGEN, Applied Biosystems, Bio-Rad, Roche, and Agilent.